ABSTRACT

Ebola virus (EBOV) causes a severe hemorrhagic fever with a deficient immune response, lymphopenia, and lymphocyte apoptosis. Dendritic cells (DC), which trigger the adaptive response, do not mature despite EBOV infection. We recently demonstrated that DC maturation is unblocked by disabling the innate response antagonizing domains (IRADs) in EBOV VP35 and VP24 by the mutations R312A and K142A, respectively. Here we analyzed the effects of VP35 and VP24 with the IRADs disabled on global gene expression in human DC. Human monocyte-derived DC were infected by wild-type (wt) EBOV or EBOVs carrying the mutation in VP35 (EBOV/VP35m), VP24 (EBOV/VP24m), or both (EBOV/VP35m/VP24m). Global gene expression at 8 and 24 h was analyzed by deep sequencing, and the expression of interferon (IFN) subtypes up to 5 days postinfection was analyzed by quantitative reverse transcription-PCR (qRT-PCR). wt EBOV induced a weak global gene expression response, including markers of DC maturation, cytokines, chemokines, chemokine receptors, and multiple IFNs. The VP35 mutation unblocked the expression, resulting in a dramatic increase in expression of these transcripts at 8 and 24 h. Surprisingly, DC infected with EBOV/VP24m expressed lower levels of many of these transcripts at 8 h after infection, compared to wt EBOV. In contrast, at 24 h, expression of the transcripts increased in DC infected with any of the three mutants, compared to wt EBOV. Moreover, sets of genes affected by the two mutations only partially overlapped. Pathway analysis demonstrated that the VP35 mutation unblocked pathways involved in antigen processing and presentation and IFN signaling. These data suggest that EBOV IRADs have profound effects on the host adaptive immune response through massive transcriptional downregulation of DC.

IMPORTANCE This study shows that infection of DC with EBOV, but not its mutant forms with the VP35 IRAD and/or VP24 IRAD disabled, causes a global block in expression of host genes. The temporal effects of mutations disrupting the two IRADs differ, and the lists of affected genes only partially overlap such that VP35 and VP24 IRADs each have profound effects on antigen presentation by exposed DC. The global modulation of DC gene expression and the resulting lack of their maturation represent a major mechanism by which EBOV disables the T cell response and suggests that these suppressive pathways are a therapeutic target that may unleash the T cell responses during EBOV infection.

INTRODUCTION

Filoviruses cause a severe hemorrhagic fever in humans and nonhuman primates with a mortality in humans of up to 90% (1). Filoviruses include five species, Zaire ebolavirus, Bundibugyo ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus, and Reston ebolavirus, belonging to the genus Ebolavirus, and a single species, Marburg marburgvirus, belonging to the genus Marburgvirus (2). Filovirus outbreaks occur in Central Africa regularly; in 2012, four filovirus outbreaks occurred in Uganda and the Democratic Republic of the Congo (3), and a new outbreak with a previously unknown clade of Ebola virus (EBOV), which belongs to the species Zaire ebolavirus, started in Guinea in the winter of 2013-2014 (4) and spread to Liberia, Sierra Leone, Nigeria, Senegal, Mali, Spain, the United States, and the United Kingdom (5). Because of the extremely high mortality of the disease caused by filoviruses and the lack of approved vaccine and treatments, work with these viruses is performed under the biosafety level 4 (BSL-4) biocontainment.

EBOV causes a severe immunosuppression in both humans and nonhuman primate models, characterized by a deficient T cell response, lymphopenia, and T cell apoptosis, despite the lack of infection of T cells (6–12). On the other hand, dendritic cells (DC), which are the most effective antigen-presenting cells and are infected by EBOV, do not undergo normal maturation despite the infection (13–15). Because DC play a key role in initiation of the adaptive immune response by processing viral antigens and presenting them to naive lymphocytes, the deficient T cell response may be linked to the deficient and/or aberrant stimulation of T cells by the infected DC.

EBOV has two proteins, VP35 and VP24, which antagonize the innate immune response. VP35 was demonstrated to antagonize the response by multiple mechanisms. Three of these mechanisms were linked to the C-terminal domain of the protein, specifically amino acids R305, K309, K319, R322, and, most importantly, R312. First, VP35 binds to IκB kinase ε (IKKε) and TANK-binding kinase I (TBK-I) and blocks their ability to phosphorylate interferon regulatory factor 3 (IRF-3) and therefore its nuclear accumulation and the resulting induction of type I interferon and type I IFN-stimulated genes (16–21). Second, VP35 dimers bind double-stranded RNA (dsRNA), which is formed as a replicative intermediate of negative-stranded viruses, including EBOV. The binding occurs in a manner similar to the binding of retinoic acid inducible gene I (RIG-I), an important sensor for RNA viruses, to dsRNA. As a result, VP35 prevents the effective engagement of RIG-I by dsRNA and thus blocks the RIG-I-mediated cascade of intracellular events resulting in induction of type I IFN and type I IFN-stimulated genes (19, 22, 23). Third, VP35 blocks and even reverses activation of dsRNA-dependent protein kinase (PKR) associated with its autophosphorylation, an effect not related to binding of VP35 to dsRNA (24, 25). In addition, VP35 antagonizes the innate immune responses by two additional mechanisms not linked to the C-terminal domain. First, VP35 inhibits IFN transcription in DC by blocking the activity of IRF-7. The blockade results from the conjugation of small ubiquitin-like modifier (SUMO) protein to IRF-7 (26). Second, VP35 inhibits an important antiviral protective mechanism, RNA interference, by interacting with components of the RNA-induced silencing complex (RISC). Specifically, VP35 was demonstrated to interact with the two dsRNA-binding proteins of the complex, trans-activation response RNA-binding protein (TRBP) and protein activator of PKR (PACT) (27). However, a separate study demonstrated that the suppression of RNA silencing by VP35 is linked to the dsRNA binding domain located in the C terminus of the protein (28).

The other EBOV protein that antagonizes the innate immune response, VP24, acts by mechanisms distinct from those of VP35. VP24 interacts with karyopherin α nuclear transporters to prevent translocation of STAT1 to the nucleus (29–32) and also binds to heterogeneous ribonuclear protein complex C1/C2 (hnRNP C1/C2), which normally interacts with karyopherin α1, and partially alters the nuclear transport of hnRNP C1/C2 (33). The interaction of VP24 with karyopherin α1 is completely reversed by the mutation K142A (29). VP24 also blocks the phosphorylation of p38 (34), which triggers the phosphorylation of transcription factors mediating the IFN response (35, 36).

In a previous study, we used recombinant fully infectious mutated live viruses in a BSL-4 biocontainment facility to test the effects of innate response antagonizing domains (IRADs) of EBOV VP35 and VP24 on maturation of human DC. We found that the EBOV VP35 and VP24 cooperatively suppress maturation of infected human DC and that the mutations R312A in VP35 and, to a lesser degree, K142A in VP24 reverse this inhibition of maturation (15).

Because of the multiple mechanisms by which VP35 and VP24 antagonize the innate immune response, it was important to evaluate the effect of the proteins and their IRADs on global gene expression in human DC. Here we tested the effects of three recombinant EBOVs carrying mutation R312A in VP35, mutation K142A in VP24, or both, on the transcription of the viral genes, on the total gene expression in infected DC by deep sequencing, and on the expression of 15 type I and type III IFN genes for up to 120 h by quantitative reverse transcription (qRT)-PCR (Fig. 1). We demonstrated a dramatic suppressive effect of VP35 on expression of multiple genes. We also demonstrated an unexpected role of the VP24 IRAD: early, the protein reduced the effects of the VP35 IRAD by increasing expression of genes, including those affected by VP35 IRAD, but later, it antagonized their expression. The pathway analysis revealed the disabling effect of the VP35 IRAD on both pro- and antiapoptotic, Toll-like receptor (TLR), and RIG-I signaling pathways.

Schematic representation of the study. (Top) Genomes of viruses used for infection; mutations R312A in VP35 and K142A in VP24 are indicated by arrows, and the inserted eGFP gene is indicated by a white rectangle. (Bottom) Time points at which RNA samples were collected for deep sequencing and for quantitation of mRNA of type I and type III IFNs. Open circles indicate deep sequencing time points, and plus symbols indicate qRT-PCR time points.

MATERIALS AND METHODS

Construction of the EBOV/VP35m/VP24m double mutant.To make the recombinant EBOV carrying both the mutation R312A in VP35 and the mutation K142A in VP24 and expressing the enhanced green fluorescent protein (eGFP) gene, we used the previously constructed plasmid pEBOV-eGFP/VP35-R312A carrying the mutation in VP35 and pEBOV-eGFP/VP24-K142A with the mutation in VP24 and each expressing the eGFP gene inserted between the NP and VP35 genes (15). The SalI-SacI fragment of the full-length pEBOV-eGFP/VP35-R312A plasmid, including part of the GP gene, the VP30 gene, and part of the VP24 gene, was replaced with its mutagenized copy from pEBOV-eGFP/VP24-K142A plasmid. The EBOV/VP35m/VP24m virus was recovered as previously described (15) and propagated in Vero-E6 monolayers. Work with the EBOV full-length clone was performed in a laboratory approved by the NIH Recombinant DNA Advisory Committee. Recovery of the virus and all work with EBOV was performed in the BSL-4 facility of the Galveston National Laboratory.

Quantitation of EBOV in DC aliquots.Aliquots of supernatants of infected DC were titrated in monolayers of Vero-E6 cells in 24-well plates and incubated at 37°C for 3 days, and plaques positive for eGFP were counted under a UV microscope.

Infection of DC and monocytes.Immature DC harvested on day 7 or monocytes were counted using trypan blue staining. Approximately 2.5 × 105 viable cells were infected with wild-type (wt) EBOV, EBOV/VP24m, EBOV/VP35m, or EBOV/VP35m/VP24m at a multiplicity of infection (MOI) of 2 PFU/cell or were mock infected. Cells were cultured in 1 ml of medium containing 10% human serum (Gemini Bio-Product, West Sacramento, CA) in 24-well plates and harvested at 8, 24, 48, 72, 96, and 120 h postinfection. Alternatively, cells were treated with Escherichia coli O55:B5 lipopolysaccharide (LPS) (Sigma-Aldrich, Saint Louis, MO) at 1 μg/ml. The statistical significance of the differences in the viral titers in supernatants of cells infected with wt EBOV and mutated EBOV were determined by a paired-sample t test.

Analysis of DC by flow cytometry.To analyze infected DC for expression of eGFP encoded by the recombinant viruses by flow cytometry, most of the infected cells were collected by pipetting, and the remaining cells, which were attached to the bottoms of the plates, were collected by applying staining buffer (phosphate-buffered saline [PBS] containing 2% fetal bovine serum and 2 mM EDTA). Cells were pelleted by centrifugation at 200 × g at 4°C for 5 min, buffer was removed, and cells were washed with the staining buffer and resuspended in 350 μl of the same buffer. Data were acquired using a FACSCanto II flow cytometer (BD Biosciences) located in the BSL-4 facility of the Galveston National Laboratory. To analyze expression of alpha interferon (IFN-α), DC were infected with wt EBOV, EBOV/VP24m, or EBOV/VP35m at an MOI of 2 PFU/cell or were mock infected. After 24 h of incubation, brefeldin A (Sigma-Aldrich, St. Louis, MO) was added at 10 μg/ml in order to inhibit IFN secretion, and cells were incubated for an additional 4 h. Thereafter, DC were stained with Live/Dead Fixable Far Red dead cell stain (Invitrogen) to discriminate between live and dead cells, washed twice with phosphate-buffered saline containing 2% human serum, and fixed and permeabilized with a fixation/permeabilization kit (BD Biosciences) per the manufacturer's instruction. Cells were then stained with antibodies specific for IFN-α2b labeled with phycoerythrin (BD Biosciences, San Diego, CA) or antibodies specific for IFN-α labeled with phycoerythrin (Miltenyi Biotec, San Diego, CA). Data were acquired using a FACSCanto II flow cytometer (BD Biosciences) in the BSL-4 facility and/or inactivated by formalin treatment according to the approved standard operating procedure, taken out of BSL-4, and analyzed using a LSRII Fortessa flow cytometer (BD Biosciences). The data were analyzed using FlowJo 7.6.1 software (TriStar, Ashland, OR). The statistical significances of the differences in the percentages of IFN-α-positive DC infected with different viruses were evaluated by a paired-sample t test.

RNA isolation.Harvested DC were washed with PBS, pelleted, and used for isolation of total RNA by TRIzol (Life Technologies, Carlsbad, CA) according to the manufacturer's recommendations. To further purify the samples, RNA was precipitated by NaCl and ethanol, resuspended in 0.3 ml of Tris-EDTA (TE) buffer, subjected to phenol extraction to remove residual proteins, precipitated as described above, and washed with 1 ml of 70% ethanol. Thereafter, RNA samples were repelleted by centrifugation at 12,000 × g, at 4°C for 5 min, air-dried, and resuspended in 50 μl of sterile RNase-free water. The final RNA solution was stored at −80°C until used for deep sequencing.

Library construction.Library construction was performed using an Illumina TruSeq RNA sample preparation v2 kit under conditions prescribed by the manufacturer (Illumina, San Diego, CA). Briefly, poly(A)-containing mRNA was purified using poly(T) oligonucleotide-attached magnetic beads, and the mRNA was eluted and fragmented by incubation at 94°C for 8 min in 19.5 μl of fragmentation buffer (Illumina). First- and second-strand synthesis, adapter ligation, and amplification of the library were performed according to the manufacturer's protocol. Samples were tracked using the “index tags” incorporated into the adapters. Library quality was evaluated using an Agilent DNA-1000 chip on an Agilent 2100 bioanalyzer. Quantification of library DNA templates was performed using qPCR and a known-size reference standard.

High-throughput sequencing.Cluster formation of the library DNA templates was performed using a TruSeq PE Cluster kit v3 (Illumina) and an Illumina cBot workstation under conditions recommended by the manufacturer. Paired-end 50-base sequencing by synthesis was performed using a TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1000 sequencing system according to protocols defined by the manufacturer. The samples (20 total) were sequenced on four lanes of an Illumina HiSeq 1000. Cluster density per lane was 820 to 940 k/mm2, and post-filter reads ranged from 148 to 218 million per lane. Base call conversion to sequence reads was performed using the Illumina software CASAVA-1.8.2. After the reads were separated by index, there was an average of 39 million read pairs per sample.

Transcriptome analysis.The reads, in fastq format, were aligned to the human hg19 UCSC reference genome (obtained from the Illumina, Inc., iGenome website). TopHat2 (version 2.0.2) (37) was first used to build a transcriptome index from the hg19 reference and the associated transcript annotation file in gene transfer format (GTF). TopHat2 splice-aware read alignment was then run with the default parameters except that the –M and –no-novel-juncs options were set. The –M option tells the program to first align the reads against the genome to filter out multihit reads and the –no-novel-juncs option tells the program to consider only known splice junctions. The transcriptome index was provided for the initial alignment of the multihit filtered reads as described in the TopHat manual.

Analysis of differential expression.The GFOLD program (38) was used to quantify and compare gene expression levels between the mock-infected sample and each of the infected samples. GFOLD was written specifically to address comparisons between samples when replicates are difficult to obtain. GFOLD considers the read counts and fold changes and outputs a GFOLD value that can be used as a reliable log2-fold change value. GFOLD version 1.0.7 was used under the default conditions. Heat maps and Venn diagrams were created using gplots (39) in R (40). Gene lists were obtained from the gene ontology database (41), release version go_201302. Gene ontology identifiers (ids) were GO:0002253 (activation of immune response), GO:005125 (cytokine activity), GO:0019221 (cytokine-mediated signaling pathway), and GO:0019882 (antigen processing and presentation).

Pathway analysis.Pathway analysis was performed with the gene set analysis software GAGE version 2.14.0 (42), which is optimized for small sample sets. About a dozen enriched KEGG (43) pathways were identified for each condition using a false discovery rate cutoff of 1%. We chose several that were high ranking and common to most of the samples to analyze further. These included Toll-like receptor signaling pathway (KEGG accession no. hsa04620), RIG-I-like receptor signaling pathway (KEGG accession no. hsa04622), and apoptosis (KEGG accession no. hsa04210). We used Pathview (44) version 1.4.0 software to visualize relative gene expression levels on the native KEGG view graphs. Pathview allows the visualization of relative expression levels of multiple samples on the same graph.

Analysis of IFN subtypes by qRT-PCR.cDNA was prepared in accordance with the manufacturer's instructions for reverse transcription of RNA using a Verso cDNA synthesis kit (Thermo Scientific, Rockford, IL). RNA primers were a mixture of random hexamers and anchored oligo(dT) primers, and the run conditions were as follows: 42°C for 30 min, 95°C for 2 min, 4°C until the end of the run. Samples were subsequently treated with RNase H (New England BioLabs, Ipswich, MA) according to the manufacturer's instructions using the following run conditions: 37°C for 20 min, 65°C for 20 min, 4°C until the end of the run. The expression of types I and III IFN was measured using the qRT-PCR method described previously (45). Briefly, 384-well assay plates were prepared by adding primers and probes with the Solo automated multichannel pipettor (Hudson Robotics, Inc., Springfield, NJ), dried, and stored in the dark at 4°C until use. TaqMan Fast Universal master mix and the primer/probe set for SDHA (succinate dehydrogenase complex, subunit A) were purchased from Applied Biosystems (Foster City, CA). Primers for the IFN transcripts and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were synthesized by the Facility for Biotechnology Resources (FBR) at the Center for Biologics Evaluation and Research (Bethesda, MD). Linear and molecular beacon probes were synthesized by FBR, and locked amino acid (LNA) probes were synthesized by Sigma-Aldrich. All primers and probes were purified by high-performance liquid chromatography (HPLC). Matrix multichannel pipettes (Thermo-Fisher Scientific) were used to add the sample cDNA, housekeeping gene primer/probe sets, standards, and master mix/water mixtures to each well. Standard curves of linearized plasmid DNA containing the IFN sequences and nontemplate controls were also included in the assay. The total volume of each PCR was 7.5 μl (3.75 μl PCR master mix, 2.25 μl primer/probe sets, and 1.5 μl cDNA template). Plates were centrifuged, mixed with a MixMate two-dimensional plate vortexer (Eppendorf, Westbury, NY), and then centrifuged again. qRT-PCR was performed using a ViiA7 real-time PCR system (Life Technologies, Grand Island, NY) under the following run conditions: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 25 s and 59°C for 1 min. qRT-PCR CT data were analyzed with ViiA7 software (Life Technologies) and input into a Microsoft Excel spreadsheet designed in-house for calculation of expression as a function of a reference gene (ΔCT) and copy numbers of each template. Target gene transcripts were normalized to micrograms of RNA input per well.

RESULTS

A recombinant EBOV with disabled IRADs in both VP35 and VP24 is viable.We previously used recombinant EBOV constructs expressing eGFP from an added gene to demonstrate that disabling the VP35 IRAD, but not the VP24 IRAD, greatly reduces the percentages of infected cells and concentration of the virus in supernatants, suggesting that the VP35 IRAD greatly promotes viral replication. In contrast, disabling the VP24 IRAD did not alter replication of the virus and increased the percentages of the infected cells. However, mutations in each of the two IRADs effectively unblocked the secretion of cytokines and chemokines by infected DC, suggesting that VP35 and VP24 each contribute to suppression of the innate immune response and that full suppression of DC maturation requires a combination of the effects of the two IRADs (15). Since the two IRADs act in EBOV-infected cells together, we first tested whether a viable replication-competent virus with both IRADs disabled can be recovered (Fig. 1). Indeed, a combination of the mutations disabling the VP35 and VP24 IRADs resulted in a recoverable, viable, and genotypically stable virus (EBOV/VP35m/VP24m) with no reversions detected by sequencing of the genome.

The VP35 IRAD has a dominant host phenotypic effect over the VP24 IRAD.We next evaluated the combined effects of the VP35 and VP24 IRADs on viral replication and protein expression in human DC. Human immature monocyte-derived DC were infected with wt EBOV expressing eGFP or its derivative carrying the mutations in VP35, VP24, or both at an MOI of 2 PFU/cell (in this and other experiments, the MOI was based on virus titration in Vero-E6 cells). Consistent with our previous report, both EBOV/VP35m and, to a lesser degree, EBOV/VP24m, but not wt EBOV, induced the formation of homotypic DC clusters (15), which represent a close correlate of the state of DC maturation (46, 47). The double mutant induced the formation of DC clusters, which looked similar to that induced by EBOV/VP35m (Fig. 2A). Our previous study demonstrated that disabling the VP35 IRAD reduces the viral protein expression and viral replication in infected DC, while disabling of the VP24 IRAD increases the protein expression but does not have any effect on viral replication (15). Testing of the double mutant demonstrated the reduced protein expression, as evidenced by the expression of eGFP (Fig. 2B and C) and the reduced replication kinetics at a level intermediate between that of EBOV/VP35m and EBOV/VP24m (Fig. 2D). Taken together, these data suggest that the VP35 IRAD has a dominant effect on protein expression and viral replication over the VP24 IRAD.

(A) Formation of homotypic DC clusters following infection with the mutated EBOV (MOI = 2 PFU/cell) on day 4 postinfection (low magnification). (B) Bright-field (top) and UV (bottom) microscopy of DC infected with wt EBOV or mutated EBOV viruses (MOI = 2 PFU/cell) on day 4 postinfection (high magnification). (C) Analysis of eGFP expression in DC infected with the indicated viruses (MOI = 2 PFU/cell) by flow cytometry on days 2 and 4. (D) Growth kinetics of the indicated viruses in DC. Suspensions of DC in triplicate were infected and washed, and aliquots of the viruses were quantitated in Vero-E6 cell monolayers by plaque titration. Titers of mutated viruses that were significantly different (P < 0.05) from wt EBOV titers are indicated by asterisks. Titers of EBOV/VP35m/VP24m were significantly higher (P < 0.05) than those of EBOV/VP35m at all time points except day 5.

EBOV induces a weak gene expression in infected DC.To evaluate the effects of the individual VP35 and VP24 IRADs or their combination on global gene expression, multiple aliquots of human monocyte-derived DC from two donors (donors 1 and 2) at 1.2 × 106 cells were infected with wt EBOV, EBOV/VP35m, EBOV/VP24m, or EBOV/VP35m/VP24m at an MOI of 2 PFU/cell or were mock infected and incubated at 37°C. The cells were harvested at 8, 24, 48, 72, 96, and 120 h. RNA from the 8- and 24-h samples were fragmented and subjected to deep sequencing (Fig. 1). The 8-h and 24-h wt EBOV-infected DC deep sequencing data were used to characterize the global gene expression (Fig. 1). For comparison, DC from two other donors (donors 3 and 4) were treated with lipopolysaccharide (LPS), and their RNA was subjected to deep sequencing. We found that at 8 h only a small number of genes were upregulated in EBOV-infected DC compared to mock-infected DC, and this number of genes increased by 24 h. In contrast, much greater numbers of genes were upregulated in response to LPS treatment (Table 1). Similarly, almost no gene downregulation was detected at 8 h after infection with wt EBOV. Thus, EBOV fails to strongly modulate gene expression, which is consistent with the previous observations on the lack of maturation of EBOV-infected cells (13–15).

Total numbers of transcripts affected by infection with wt EBOV or the mutated viruses or by treatment with LPSa

The VP35 IRAD blocks host gene expression.In contrast to wt EBOV, infection with EBOV/VP35m induced multiple genes at 8 h postinfection, with many genes upregulated more than 64-fold over baseline (Table 1). The trend continued at 24 h, with the numbers of genes in EBOV/VP35m-infected cells expressed at a high level greatly exceeding that in wt EBOV-infected cells. A small number of genes were downregulated at 8 h in response to infection with EBOV/VP35m, but not wt EBOV, but a greater number of genes were downregulated in both groups by 24 h. Nevertheless, the number of downregulated genes in DC infected with EBOV/VP35m was lower than the number of upregulated genes, especially at 8 h. These data suggest that the VP35 IRAD effectively blocks gene expression in infected DC.

The VP24 IRAD induces host gene expression at 8 h but blocks it at 24 h.Unexpectedly, the number of genes upregulated in DC infected with EBOV/VP24m at 8 h was lower than that in wt EBOV-infected cells but by 24 h, this number increased and exceeded that in wt EBOV-infected cells (Table 1). Taken together, these data suggest that the VP35 IRAD blocks gene expression early but the VP24 IRAD demonstrates the blocking effect late.

The different effects of the VP35 and VP24 IRADs on expression of cellular genes do not result from the different levels of expression of the viral genes by the mutated viruses.The dramatic upregulation of expression of host genes associated with the disruption of the VP35 IRAD can in principle result from enhanced expression of EBOV proteins in EBOV/VP35m-infected cells, compared to that in wt EBOV-infected cells. Even though the number of viral particles in EBOV/VP35m-infected cells was greatly reduced (Fig. 2D), it can be affected by viral budding and therefore may not necessarily reflect the abundance of the viral transcripts in infected cells. To determine the abundance of viral mRNA in infected cells, we quantitated the numbers of reads for each gene (Fig. 3A and B). The mean base coverage depth of all (NP-eGFP, eGFP-VP35, VP40-GP, VP30-VP24) intergenic regions ranged between 1% and 10% of that for the NP gene reads in the same samples, suggesting that most of the reads resulted from mRNAs, and only a small fraction of them was generated from the viral genome and possibly readthrough transcripts. Quantitation of the wt EBOV reads demonstrated that the pattern of transcription of the viral genome generally followed the gradient of transcription from the 3′ to the 5′ end of the genome associated with transcription attenuation at gene junctions originally described for vesicular stomatitis virus (48, 49). Even though the gradient of transcription is believed to be universal for nonsegmented negative-strand viruses (50), the levels of transcription of individual genes may not necessarily follow the gradient exactly due to the regulatory role of different elements of gene junctions, which may be different for different pairs of genes. The elements, which effect transcription, include intergenic regions and transcription termination signals, as demonstrated for respiratory syncytial virus (51, 52) and recently for EBOV (53). In our study, the abundance of the eGFP mRNA was lower than that of the downstream genes at both time points. Furthermore, at 24 h, the abundance of VP40, GP, and VP30 was greater than that of NP. These differences among expression of EBOV genes may reflect not only the efficiency of transcription but also the different stability of each mRNA. Analysis of the effects of the VP35 and VP24 mutations demonstrated three effects. First, introduction of either the VP24 or VP35 mutation reduced the level of mRNAs in infected cells. Second, the combination of the two mutations increased rather than reduced the levels of transcripts at 8 h but not at 24 h. Third, introduction of the mutations modified the levels of transcription of individual genes; the most obvious effect observed was the relative reduction of the VP40, GP, and VP30 transcripts, with somewhat lesser effects on other transcripts. Importantly, even though at 8 h introduction of the mutation in VP35 reduced the levels of viral mRNA by 2.5- to 4.1-fold, levels of the most analyzed host mRNA in infected cells were strongly increased rather than decreased (Table 1). These data suggest that the observed changes in host gene expression associated with the mutations in EBOV do not result from the different levels of expression of the viral genes by the mutated viruses. However, we do not exclude that changes in the levels of viral mRNA associated with the mutations could have a modest effect on the level of host gene expression. To determine if the observed viral gene expression effects are specific for DC or can be observed in other types of cells, we repeated the experiment with human monocytes, which were analyzed at 24 h. We found that the pattern of the relative abundance of individual mRNA transcripts was generally similar to that in DC, with the greatest level of transcripts detected for VP40 and GP (Fig. 3C). However, unlike the findings in infected DC, introduction of the VP35 or VP24 mutations did not reduce the levels of viral mRNA in infected monocytes, which may suggest a lower level of IFN response in the latter cells. The overall conclusion from this analysis is that the dramatically increased expression of the host's genes in cells infected with EBOV/VP35m, compared to wt EBOV, is not a consequence of an increased abundance of viral mRNA, which was reduced rather than increased.

Quantitative analysis of viral transcripts, including the eGFP transcript, in DC (A and B) or monocytes (C) from two donors (left and right panels) infected with the indicated viruses (MOI = 2 PFU/cell) at 8 h (A) and 24 h (B and C) postinfection. The numbers of viral mRNA reads were normalized to the number of all reads in the sample library and normalized to the length of the transcripts to show the relative abundance of each transcript. For GP, the length of the full-length edited mRNA encoding GP1/2 was used for normalization. One arbitrary unit is equal to 100 reads after the normalizations.

The IRADs in VP35 and VP24 suppress different sets of genes.Disabling of IRADs in VP35 and VP24 resulted in upregulation of sets of genes, which overlapped only partially (Fig. 4A). At 24 h postinfection, the numbers of genes upregulated more than 16-fold by both EBOV/VP35m and EBOV/VP24m were 113 (donor 1) and 69 (donor 2; data not shown), which are 43% and 30%, respectively, of the total number of genes upregulated by EBOV/VP35m and 84% and 81%, respectively, of the number of genes upregulated by EBOV/VP24m. We next calculated upregulation of genes belonging to the following three families: (i) genes encoding activation of immune response, defined as any process that initiates an immune response, (ii) genes encoding cytokine activity, defined as functions to control the survival, growth, differentiation, and effector function of tissues and cells, and (iii) genes encoding the cytokine-mediated signaling pathway, defined as a series of molecular signals initiated by the binding of a cytokine to a receptor on the surface of a cell, and ending with regulation of a downstream cellular process, e.g., transcription (Fig. 4B). We calculated the numbers of genes that were upregulated at 24 h by both EBOV/VP35m and EBOV/VP24m at least 4-fold and as percentages of genes upregulated by either EBOV/VP35m or EBOV/VP24m. The respective percentages for EBOV/VP35m in donor 1 (Fig. 4B) and donor 2 (data not shown) for the three groups of genes were 69% and 44%, 93% and 44%, and 96% and 60%. The respective percentages for EBOV/VP24m were 88% and 87%, 70% and 67%, and 91% and 94%. These data suggest that VP35 and VP24 suppress the innate immune response by different mechanisms, which overlap only partially, and that VP35 is likely to have a greater suppressing effect.

VP35 and VP24 IRADs suppress different sets of genes. (A) Venn diagrams of the mRNAs upregulated in DC from donor 1 in response to infections with wt EBOV or the mutated EBOV. The fold upregulation is indicated at the left. (B) Venn diagrams of the mRNAs belonging to the indicated functional groups, which were upregulated above 4-fold in DC from donor 1 in response to infections with wt EBOV or the mutated EBOV.

The IRADs in VP24 and VP35 render opposite effects on many genes.To determine the mutual effects of VP35 and VP24, we compared the numbers of genes upregulated separately by EBOV/VP35m or EBOV/VP24m with that upregulated by EBOV/VP35m/VP24m. Surprisingly, we found that at 8 h, when a strong upregulation of gene expression was demonstrated for EBOV/VP35m, but not EBOV/VP24m, the number of genes upregulated by EBOV/VP35m/VP24m was much below that upregulated by EBOV/VP35m (Table 1; Fig. 4A). At 24 h postinfection, when the suppressive effect of VP24 was evident, the number of genes upregulated by the double mutant was slightly greater than that upregulated by EBOV/VP35m. However, the combined numbers of genes upregulated by the two individually mutated viruses, EBOV/VP35m and EBOV/VP24m, were far greater (Table 1; Fig. 4A). Taken together, these data suggest that the VP35 IRAD effectively suppresses expression of multiple genes both early and late, but the VP24 IRAD stimulates some of them, especially early, and the two IRADs change the expression of many genes in opposite directions.

The IRADs in VP35 but not VP24 suppress the expression of markers of DC maturation.To focus on the effects of EBOV and the IRADs in VP35 and VP24 on genes whose expression correlates with DC maturation, we compared the levels of mRNA of CD38, CD40, CD40 ligand, CD80, CD83, CD86, ICAM1, and multiple HLA class I and class II antigens (Fig. 5). At 8 h, infection with EBOV/VP35m resulted in a very strong upregulation of CD38 and weaker (2- to 4-fold, compared to the level in wt EBOV-infected DC) upregulation of CD40, CD80, CD83, and HLA-F in DC from both donors and of ICAM1 in DC from donor 1. None of these genes were affected by wt EBOV or EBOV/VP24m. Interestingly, expression of CD38 in DC infected with EBOV/VP35m/VP24m was strongly reduced compared to the level in EBOV/VP35m-infected cells, suggesting a stimulatory role of the VP24 IRAD on expression of this gene.

At 24 h postinfection, wt EBOV induced a >4-fold increase in the levels of mRNA of CD38, CD80, and CD83 in DC from both donors and also CD40 in DC from donor 1. We also detected a >2-fold reduction in the level of class I HLA-DOA in DC from donor 1; in cells from donor 2, the reduction was less pronounced. As at 8 h postinfection, EBOV/VP24m did not change the expression of the tested genes, while infection with EBOV/VP35m strongly increased the levels of CD38 mRNA in DC from both donors. Infection with EBOV/VP35m also increased, 2- to 4-fold above the levels in wt EBOV-infected DC, the levels of CD40, CD80, and CD83 in cells from both donors and class I HLA-A and HLA-B in cells from donor 2. Infection with EBOV/VP35m/VP24m resulted in effects similar to those of EBOV/VP35m. These data demonstrate that VP35, but not VP24, blocks the expression of DC maturation markers.

The VP24 IRAD counteracts the VP35-mediated suppression of cytokine expression early but not late.We next determined the effect of the virus and the IRADs in VP35 and VP24 on the expression of 81 cytokines in infected DC (Fig. 6). We found that at 8 h, wt EBOV did not induce any of the cytokines (above a 4-fold level), with the exception of IL-1B (in both donors) and IL-1A, IL-6, and TNFSF10 (in one donor each). In contrast, EBOV/VP35m upregulated 10 cytokines in both donors. The most dramatic upregulation was detected for IL-27 (112- and 29-fold compared to wt EBOV levels in donors 1 and 2, respectively), IL-6 (108- and 9-fold), and TNFSF10 (29- and 25-fold, respectively). In contrast, no effect of EBOV/VP24m on the expression of cytokines was detected. The presence of the VP24 mutation in the double mutant not only did not increase the expression of any gene (above 4-fold), compared to the level in EBOV/VP35m-infected DC, but in fact reduced the expression of several genes. For example, a >2-fold reduction, compared to that of EBOV/VP35m, was detected for IL-1RN, IL-27, IL-6, and IL-7 in both donors and for IL-15, IL-27, IL-29, TNFSF10, and TNFSF13B in one donor, with a lesser reduction observed in another donor.

At 24 h postinfection, wt EBOV upregulated several genes above the 4-fold level in both donors. Similar to the results at the 8-h time point, infection with EBOV/VP35m resulted in upregulation of multiple genes above the wt EBOV levels, most of which were also upregulated at the 8-h time point. No genes were upregulated by EBOV/VP24m, compared to wt EBOV. Infection with EBOV/VP35m/VP24m resulted in levels of gene expression comparable to those of EBOV/VP35m, with one gene, the IL-12A gene, upregulated to a >4-fold-greater level than EBOV/VP35m in both donors; two genes, the IL-36RN and VEGFC genes, were upregulated in DC from one donor only above the EBOV/VP35m level. Similar to the results at the 8-h time point, no increase in gene expression, compared to the level in wt EBOV-infected DC, was detected in cells infected with EBOV/VP24m at 24 h postinfection.

Interestingly, at 24 h postinfection, some genes were downregulated >2-fold, compared to mock-infected cells. Specifically, infection with wt EBOV and EBOV/VP24m reduced the expression of IL-16, and infection with EBOV/VP24m reduced the expression of IL-18 and VEGFB, compared to mock-infected DC, in cells from one of the two donors. Furthermore, infection with EBOV/VP35m and EBOV/VP35m/VP24m resulted in >2-fold reduction, compared to mock-infected DC, of expression of TGFB2 in both donors, VEGFB in donor 1, and IL-16 in donor 2. Taken together, these data suggest that early (at 8 h), but not late (24 h), the VP24 IRAD counteracts the VP35-mediated suppression of cytokine expression.

Effect of IRADs in VP35 and VP24 on expression of chemokines and chemokine receptors.Next, the effects of VP35 and VP24 IRADs on mRNA of chemokines (Fig. 7A) and chemokine receptors (Fig. 7B) were analyzed. At 8 h postinfection, wt EBOV upregulated only a few chemokines >4-fold. Infection with EBOV/VP35m resulted in a >4-fold increase, compared to wt EBOV, in the levels of several CCL and CXCL chemokines. In contrast, EBOV/VP24m induced mRNA of several chemokines at a level >2-fold below that in wt EBOV-infected cells. Infection with EBOV/VP35m/VP24m induced the same chemokines as EBOV/VP35m but at lower levels. For some chemokines, the level of expression in DC infected with the double mutant was >2-fold lower than that in DC infected with EBOV/VP35m: CXCL-9, CXCL-10, and CXCL-11 in DC from both donors and CCL-8 in donor 2 only. At 24 h postinfection, wt EBOV upregulated mRNA of multiple chemokine genes. Similar to the results at the 8-h time point, infection with EBOV/VP35m resulted in a strong upregulation of multiple genes, compared to wt EBOV. CCL-24 and CCL-26 transcripts were reduced in DC from one of the two donors. Infection with EBOV/VP24m did not change the level of mRNA, compared to wt EBOV, of most of the chemokines, with the exception of an increase in CXCL-9 in DC from both donors, an increase in CCL-8 in DC from donor 1, and a reduction in CXCL-6 in DC from donor 2. Infection with EBOV/VP35m/VP24m induced mRNA of a set of genes similar to that induced by EBOV/VP35m; however, several genes were transcribed at a >2-fold-lower level than that in EBOV/VP24m-infected DC: CCL-15, CXCL-5, and CXCL-6 transcripts in DC from both donors and CXCL-16 transcripts in DC from donor 1.

Levels of mRNA of chemokine receptors was much less affected by the viruses, compared to chemokines. At 8 h postinfection, none of the receptors were upregulated by wt EBOV, and two genes were downregulated >2-fold in the mutated viruses, compared to wt EBOV. Specifically, we detected >2-fold downregulation of CCR-6 in DC from donor 1 infected with EBOV/VP35m and DC from both donors infected with EBOV/VP35m/VP24m and of CXCR4 in DC from donor 1 infected with EBOV/VP24m. At 24 h postinfection, >4-fold upregulation of CCR-5 was detected in wt EBOV-infected DC from donor 1, and a strong upregulation of CCR-7 and CXCR-7 in DC was seen in both donors infected with EBOV/VP35m and EBOV/VP35m/VP24m. Taken together, these data demonstrate that the VP35 IRAD blocks expression of multiple chemokines, but the VP24 IRAD counteracts the VP35-mediated block early (at 8 h) but not late (at 24 h). They also demonstrate that in contrast to chemokines, the two IRADs have a minimal effect on expression of chemokine receptors.

Effect of IRADs on kinetics of induction of multiple IFNs.We analyzed the effects of the VP35 and VP24 IRADs on the induction of multiple IFNs belonging to types I, II, and III (Fig. 8). At 8 h postinfection, no consistent upregulation of IFNs by wt EBOV was detected. In contrast, in cells infected with EBOV/VP35m, >4-fold upregulation of IFN-α1, -α2, -α8, -α13, -α14, and -λ1, but not the other IFNs, was detected. A very strong (182- and 49-fold in DC from donors 1 and 2, respectively) upregulation of IFN-β1 was detected. IFN-ω was upregulated in DC from donor 1 only, and expression of IFN-κ and IFN-ε was not altered. Unlike most of the type I IFNs, no significant upregulation of IFN-γ, which is the type II IFN, was detected. In contrast to EBOV/VP35m, infection with EBOV/VP24m did not increase the levels of mRNA for any of the IFNs tested. Moreover, the IFNs upregulated by EBOV/VP35m were not upregulated or were weakly upregulated by the double mutant, suggesting a dominant effect of VP24 at this time point. At 24 h, no induction of IFNs by wt EBOV was detected, except for IFN-β1 and -λ1. EBOV/VP35m upregulated all IFNs tested, with the exception of IFN-α6, IFN-κ, and IFN-γ. In contrast to the findings at the 8-h time point, EBOV/VP24m upregulated multiple IFN genes, i.e., the IFN-α1, -α2, -α8, -α13, -α14, and -β1 genes, in DC from donor 1 but not donor 2. The double mutant induced IFN expression to levels comparable to that induced by EBOV/VP35m. One noticeable exception was IFN-γ, which was upregulated (above 4-fold) by the double, but not single, mutants. Analysis of IFN-α/β receptors IFNAR1 and IFNAR2 and IFN-γ receptors IFNGR1 and IFNGR2 demonstrated no changes in their expression (above 4-fold).

To determine the effect of the mutations on the expression of IFNs over a longer period of time and to confirm the transcriptome data by an alternative method, we analyzed RNA samples from time points up to 120 h by qRT-PCR analysis (45) (Fig. 9). We found that IFN-α1, -α7, -α8, -α14, -β, and -λ1 mRNAs were most upregulated at multiple time points. Consistent with the transcriptome data, the effects of the mutations in VP35 and/or VP24 depended on the time point. Early, at 8 h, in comparison to the other viruses, EBOV/VP35m induced a greater amount of IFN-α1, -α2, -α7, -α8, -α14, -β, and -λ1 mRNAs, followed by the double mutant. The level of IFNs induced by EBOV/VP24m was low and was comparable to or lower than that induced by wt EBOV. At 24, 48, and 72 h, the level of most IFNs induced by EBOV/VP35m/VP24m was equal to or marginally greater than that induced by EBOV/VP35m, while that induced by EBOV/VP24m was much lower and equal to or marginally greater than that induced by wt EBOV. At 96 and 120 h, the level of most IFNs was greatest for EBOV/VP35m/VP24m, followed by EBOV/VP35m, while as at the earlier time points, IFN levels induced by EBOV/VP24m were much lower and were equal to or marginally greater than that induced by wt EBOV.

Radial plots of qRT-PCR analysis for type I and III IFN subtype expression in response to the various viruses at time points up to 120 h. Shown in log10 scale as copy number per μg of RNA. IFN-α subtypes are ordered according to a phylogenetic plot of amino acid sequence similarity.

These data suggest that VP35 effectively suppresses the induction of IFN genes early after infection and that the suppressive effect lasts for many days. In contrast, VP24 partially weakens the suppressive effect of VP35 on IFN expression early but enhances the suppressive effect later, with the most pronounced suppressive effect detected at the latest time points analyzed, 4 to 5 days postinfection.

The increased production of IFN associated with the disabling of IRADs is related to a highly activated fraction of DC.The low percentages of eGFP-positive (eGFP+) DC after infections can be explained by either low susceptibility of the whole DC population to EBOV infection or by susceptibility to infection by a subset of the DCs. To distinguish between these two possibilities, we compared the percentages of eGFP+ DC at 24 and 48 h after infection with wt EBOV at two different MOIs, 2 and 10 PFU/cell, and found that the increase in MOI only weakly or modestly increased the percentage of eGFP+ cells, with the increase being more pronounced for the mutated viruses (Fig. 10A). These data suggest that there is a subset of EBOV-susceptible DC and that the higher MOI may transiently overcome the resistance of some of the DC to infection. Interestingly, infections also modestly increased the eGFP mean fluorescence intensity (MFI) of the eGFP-low/negative (eGFPlow/−) population compared to that of mock-infected cells (Fig. 10B). Although not all mean increases were significant due to donor-to-donor variations, the increases were detected in DC from each donor. Because type I IFN mediates resistance to infection and can act in autocrine and paracrine manners, we next asked whether type I IFN expression was limited to productively infected cells. DC were infected with wt EBOV, EBOV/VP35m, or EBOV/VP24m for 24 h and the levels of IFN-α2b or the total IFN-α were determined by intracellular staining, followed by flow cytometry analysis (Fig. 10C to E). Infection with wt EBOV resulted in proportions of eGFP+ cells expressing IFN-α2b and total IFN-α equal to 0.2% and 0.6%, respectively, and much smaller proportions of eGFPlow/− cells, i.e., 0.01% and 0.08%, respectively. To analyze the effects of the mutations, we normalized the percentages of IFN-α2b+ or total IFN-α+ DC after infections with the mutated viruses to that after infections with wt EBOV (Fig. 10E). We found that the mutation in VP35 dramatically increased the percentages of IFN+ eGFP+ DC in cells from all donors. The mutation in VP24 also resulted in an increase in the percentages of IFN+ eGFP+ DC in cells from all donors, with the exception of cells from one donor that were stained with IFN-α2b-specific antibodies. The strong increases in the percentages of IFN+ DC associated with the mutation on VP35 or VP24 were also detected in eGFPlow/− populations of cells from most of the donors. Since eGFPlow/− DC in general represent a much greater proportion of cells than eGFP+ DC (Fig. 10A), these data suggest that the observed upregulation of IFN-α and most likely other IFNs (Fig. 8 and 9) associated with the mutations in IRADs are related not only to a subset of an eGFP+ highly infected population of DC but also to a subset of eGFPlow/− cells which may be infected at a low level.

Analysis of type I IFNs in eGFP+ and eGFPlow/− DC. (A) Flow cytometry analysis of eGFP expression in DC infected with the indicated viruses at an MOI of 2 or 10 PFU/cell. (Left) Primary flow cytometry data with DC from a representative donor at 48 h postinfection. The percentages of eGFP+ cells are indicated for each histogram. (Right) Percentages of eGFP+ DC after infection at an MOI of 10 PFU/ml were normalized to that after infection at an MOI of 2 PFU/cell (100%, dotted line) at 24 and 48 h postinfection. DC from individual donors are indicated by symbols, and the mean values are indicated by horizontal lines. No statistical difference between eGFP levels for infection at an MOI of 2 or 10 was found. (B) Levels of eGFP in the eGFPlow/− DC populations. (Left) eGFP expression by DC from a representative donor at 24 h after infection by wt EBOV or EBOV/VP35m at an MOI of 2 PFU/cell (black) or 10 PFU/cell (red). (Right) MFI values at 24 and 48 h after infections with the indicated viruses at an MOI of 2 or 10 PFU/cell or after mock infections; statistically significant differences in results compared to mock infections (P < 0.05) are indicated by black asterisks, and infections at an MOI of 10 PFU/cell, which resulted in IFN levels significantly increased (P < 0.05) over that for infections at an MOI of 2 PFU/cell, are indicated by red asterisks. (C to E) Expression of IFN-α by eGFP+ and eGFPlow/− DC. (C) Gating strategy used to analyze the levels of IFN-α in eGFP+ and eGFPlow/− DC. SSC-A, side scatter A; FSC-A, forward scatter A; FSCH, forward scatter height; PE, phycoerythrin. (D) Analysis of IFN-α2b in eGFP+ and eGFPlow/− populations of DC from a representative donor infected with the panel of viruses or mock infected. The percentages of type I IFN-positive cells are indicated for eGFP+ and eGFPlow/− populations. (E) Numbers of DC positive for IFN-α2b or total IFN-α after infection with the indicated mutated viruses are indicated as fold increases over wt EBOV levels. Values for DC from five donors are indicated by symbols, and mean values for each treatment are indicated by horizontal lines. Infections with the mutated viruses, which resulted in IFN levels that were significantly increased (P < 0.05) over that for wt EBOV, are indicated by asterisks.

VP35 and VP24 IRADs cause different perturbations of multiple signaling pathways involved in the induction of the adaptive and innate immune responses.We next used pathway analysis to analyze the effects of IRADs on pathways involved in the induction of the adaptive and innate immune responses. The effects of the VP35 IRAD, the VP24 IRAD, or both were compared. Analysis of the apoptotic pathways (see Fig. S1A in the supplemental material) demonstrated that at 8 h, disabling of the VP35 IRAD upregulated the expression of tumor necrosis factor alpha (TNF-α) and Fas ligand but not TNF-α-related apoptosis-inducing ligand (TRAIL), suggesting that the blockade of apoptosis starts at the death ligand level. Next, EBOV/VP35m and EBOV/VP35m/VP24m upregulated the expression of the death receptors Fas and TRAIL receptor I, the adaptor molecules tumor necrosis factor receptor type 1-associated death domain protein (TRADD), receptor-interacting protein 1 (RIP-I), and TNF receptor-associated factor 2 (TRAF2). They also upregulated the expression of the proapoptotic BCL-2 family member BID and caspase-7. On the other hand, these mutants also upregulated the antiapoptotic proteins FLICE-like inhibitory protein (FLIP), members of the BCL-2 family Bcl-2 and Bcl-XL, and inhibitor of apoptosis (IAP). Analysis of the NF-κB pathway demonstrated that EBOV/VP35m and EBOV/VP35m/VP24m upregulate both this protein and its inhibitor, IκB, which results in the upregulation of the NF-κB-induced prosurvival proteins Bcl-2, IAO, and Bcl-XL. Thus, the VP35 IRAD suppresses both the proapoptotic and prosurvival pathways.

Analysis of the Toll-like receptor signaling pathways (see Fig. S1B in the supplemental material) demonstrated that EBOV/VP35m and EBOV/VP35m/VP24m upregulate TLR1 and TLR2 and the adaptor protein MyD88 molecule, which can be upregulated by Toll-like receptors. The viruses also upregulated multiple downstream proteins, including various adaptor proteins, namely, TIR domain-containing adapter-inducing interferon-β (TRIF), TNF-associated factor 3 (TRAF3), IκB kinase epsilon (IKKε), the NF-κB and its precursor p105, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IkBα), osteopontin (OPN), IRF-7, and a transcription factor activator protein 1 (AP-1). Furthermore, the viruses upregulated inflammatory cytokines, including antiviral type I IFN, which results in the induction of proinflammatory effects and chemotaxis of immune cells, and costimulatory molecules, which are involved in stimulation of T cells. These data suggest that the VP35 IRAD inhibits multiple molecules and pathways involved in the induction of both the innate and adaptive immune responses, including those responding to stimuli other than viruses, such as TLR2.

Analysis of RIG-I-like receptor signaling demonstrated that many molecules affected by the RIG-I-like receptor signaling were upregulated due to infection with EBOV/VP35m or EBOV/VP35m/VP24m (see Fig. S1C in the supplemental material). Specifically, disabling of the VP35 IRAD resulted in upregulation of several downstream molecules, including TRAF-associated NF-κB activator (TANK), IKKε, IRF-7, KIP-1, IκB, and NF-κB, which results in upregulation of type I IFNs and inflammatory cytokines. RIG-I itself was equally upregulated in response to each of the four viruses in DC from donor 1 but more highly upregulated in response to infection with EBOV/VP35m or EBOV/VP35m/VP24m in DC from donor 2. Analysis of additional signaling pathways demonstrated that disabling of the VP35 IRAD unblocked the expression of Janus kinase (JAK) and signal tranducer and activator of transcription (STAT) (the JAK-STAT signaling pathway), and major histocompatibility complex class I (MHC-I), β2 microglobulin, and MHC-II (the antigen processing and presentation pathway) (data not shown).

DISCUSSION

DC are critical for initiation of the adaptive immune response. We previously demonstrated that introduction of single mutations in EBOV IRADs reverses the block imposed by EBOV on DC maturation and the secretion of cytokines and chemokines (15), demonstrating a mechanism by which the virus disables the adaptive immune response. Here we aimed at a comprehensive in-depth characterization of the effect of IRADs on maturation of DC by deep sequencing of their transcriptome after infection with wt EBOV or EBOV mutants in which the VP35 IRAD, the VP24 IRAD, or both were disabled by point mutations. Instead of using established cell lines or inbred animals, we designed an experimental system that we can best extrapolate toward in vivo EBOV infection that included fully infectious virus and DC derived from donor blood. Analysis of the transcriptomes in DC from two donors demonstrated remarkably similar responses (Fig. 5 to 8).

The VP35 IRAD was found to globally affect the levels of transcripts of multiple genes at both the 8- and 24-h time points, including type I and III IFN genes, which were also tested at later time points. At 8 h, disabling of the IRAD resulted in mostly upregulation of multiple genes, with far fewer genes downregulated; at 24 h, a greater number of genes was upregulated, while a substantial but much lesser number of other genes was downregulated (Table 1). The affected genes belonged to all groups analyzed: markers of DC maturation, cytokines, chemokines, chemokine receptors, and IFNs (Fig. 5 to 9). During maturation, DC modulate expression of chemokine receptors; specifically, they increase expression of CCR7 (54). The ligands for CCR7, CCL19, and CCL21 are expressed by various cells in lymphoid tissues that result in effective migration of activated DC to lymph nodes (55) and thus in activation of antigen-specific T cells (56). We previously demonstrated that the VP35 IRAD greatly inhibits the migration of DC infected with EBOV/VP35m toward a gradient of CCL19 (15). In the present study, we found that disruption of the VP35 IRAD resulted in an increase in expression of CCR7 in two donors, by 5.4- and 9.9-fold (Fig. 7B). Moreover, it also resulted in the increase in expression of CCL19 by 7.4- and 47.8-fold, respectively (Fig. 7A).

The effect of the VP24 IRAD was found to be very different from that of the VP35 IRAD both early (at 8 h postinfection) and late (24 h and later). Paradoxically, at 8 h, disabling of the VP24 IRAD resulted in the decrease of expression of many genes whose level was upregulated in response to infection with wt EBOV. This unexpected effect was observed among genes belonging to all five groups analyzed (Fig. 5 to 9). In contrast, at 24 h, disabling of the VP24 IRAD resulted in upregulation of expression of a large number of genes belonging to all groups analyzed; however, a large, although lesser, number of genes were downregulated.

To analyze the combined effect of the VP35 and VP24 IRADs, we developed a virus with both of them disabled (EBOV/VP35m/VP24m). Analysis of DC infected with this virus provided the additional evidence of the stimulating role of the VP24 IRAD at 8 h: the level of mRNA in DC infected with this virus was reduced rather than increased compared to the level of mRNA in DC infected with EBOV/VP35m (Fig. 5 to 9). The effect was observed among all groups of genes analyzed but was especially strong among the IFN gene group (Fig. 8). Thus, at the early time point, the VP24 IRAD counteracts, rather than enhances, the effect of the VP35 IRAD. Again, in contrast to the early time point, at 24 h, the number of transcripts upregulated due to the two mutations was comparable to that upregulated due to the single VP35 mutation, with some transcripts upregulated by the double mutant at a somewhat higher level and others upregulated at a somewhat lower level. Moreover, when the combined effect of the two mutations on expression of IFN genes was analyzed over a longer period of time, the levels of transcripts of almost all subtypes of IFN were the highest in DC infected with EBOV/VP35m/VP24m, followed by EBOV/VP35m (Fig. 9). Taken together, these data suggest that the effect of VP24 IRAD changes over time, paradoxically, from enhancing the expression early to suppressing it, in cooperation with the VP35 IRAD, late. We are unaware of any previously described immunomodulatory proteins of RNA viruses whose effects are time dependent. Interestingly, the VP24 and VP35 IRADs affected the expression of only partially overlapping sets of genes, and disabling both of them resulted in stimulation or suppression of a considerable number of genes not affected by each of the IRADs separately (Fig. 4). Analysis of multiple type I and III IFN subtypes over a longer period of time (up to 5 days) demonstrated remarkably similar IFN expression patterns for wt EBOV and each of the mutants, which differed mostly by the abundance of transcripts (Fig. 9). For example, while the mutations greatly unblocked the expression of all IFN genes, IFN-α1, -α8, -α14, -β, and -λ1 transcripts were most abundant for all viruses, including wt EBOV, and at all time points tested. These data are consistent with the previously established effects of IRADs on common upstream IFN regulatory factors, such as IRF-3, discussed in the introduction.

Analysis of multiple pathways affected by EBOV demonstrated only marginal effects of wt EBOV that may be consistent with the lack of DC maturation (see Fig. S1 in the supplemental material; data not shown). Infection with EBOV/VP24m also resulted in marginal effects on the pathways. In contrast, EBOV/VP35m and EBOV/VP35m/VP24m stimulated both apoptotic and antiapoptotic pathways, Toll-like receptor signaling pathways, and RIG-I-like receptor signaling pathways (see Fig. S1), suggesting that VP35 is a strong suppressor of innate immunity while the role of VP24 is more subtle. Interestingly, a recently published study demonstrated that plasmid-based delivery of VP35 to DC blocks TLR pathways only partially (57). However, unlike our work, this study did not involve infection of DC with live EBOV, which could result in greater levels of VP35 expression in infected DC.

Analysis of the total number of viral transcripts (Fig. 3) showed two unexpected findings. First, while the effects of disabling the VP35 IRAD and, to a lesser degree, the VP24 IRAD were comparable at 8 and 24 h, the viral transcripts were much less abundant at 8 h, suggesting that the low threshold level of the viral replication was sufficient to both stimulate and simultaneously disable (by the IRADs) the innate response to the infection. Second, the strong attenuation of EBOV/VP35m (Fig. 2B to D) did not always correlate with the levels of the viral transcripts in the infected cells, which were comparable between this virus and the nonattenuated EBOV/VP24m (Fig. 3). This discrepancy indicates that the increased expression of the virus-encoded eGFP, associated with the disruption of VP24 IRAD (15), is not a consequence of its effect on transcription of the viral genes but instead is related to suppression of translation or some other effect mediated by the IRAD. This discrepancy is also consistent with the strong upregulation of multiple cell transcripts by EBOV/VP35m, compared to wt EBOV, despite the dramatically reduced concentration of the former viral particles in cell medium and the reduced number of eGFP+ cells. Overall, we found no obvious dependence of expression of the host genes (Fig. 4 to 9) on the abundance of the viral transcripts (Fig. 3). One caveat is that the level of cellular transcripts analyzed in the study represents a balance of their transcription and turnover. However, the mechanistic studies with plasmid-expressed EBOV VP35 and VP24 mentioned in the introduction suggest that most of the effects of IRADs observed in the present study result from their effect on the transcription of host genes, although some turnover effects cannot be excluded.

Unexpectedly, flow cytometry analysis demonstrated that after infections, not only a fraction of eGFP+ DC but also a much smaller fraction of eGFPlow/− DC secreted IFN-α. This is consistent with the small but detectable increase in the levels of eGFP in the eGFPlow/− population following exposure to the viruses, possibly indicating a low-level infection of a fraction of these cells. In agreement with the transcriptome and qRT-PCR data (Fig. 5 to 9), the levels of IFN-α determined by flow cytometry were barely detectable in cells infected with wt EBOV but increased many times after infections with the mutated viruses (Fig. 10). The observed increase in a fraction of eGFP+ and eGFPlow/− cells apparently was sufficient for maturation of the whole population of DC, as evidenced by the levels of expression of phenotypic markers of maturation such as CD86 (15). A previous study with respiratory paramyxoviruses demonstrated that even a very low-level infection of DC is sufficient for their maturation (58). Why did virtually 100% of DC undergo maturation after exposure to the mutated EBOVs despite the fact that following infection only a fraction of them were highly positive for type I IFNs? It is possible that the elevated levels of type I IFNs and possibly some other proteins, such as cytokines and chemokines, secreted by DC that are highly activated in response to infection with the mutated viruses trigger the cascade of reactions leading to maturation of the whole population of DC. Alternatively, it is possible that cells secreting high levels of type I IFNs undergo maturation and transfer MHC-peptide complexes to cells which do not secrete type I IFNs, resulting in their maturation; previous studies have demonstrated that such transfer, known as “cross-dressing” (59, 60), induces upregulation of costimulatory molecules CD80 and CD86 by recipient cells (61). Finally, it is also possible that the induction of type I IFNs by the mutated viruses detected in our experiments (Fig. 10B to E) is very transient, and over a longer period of time, most of DC exposed to the mutated viruses undergo a brief period of high expression of type I IFNs, which is sufficient to trigger their maturation.

In summary, this study shows that the lack of maturation of EBOV-infected DC correlates with the global block in their expression, which is caused by the VP35 and VP24 IRADs. The VP35 IRAD suppresses expression of multiple genes involved in maturation of DC and the induction of the innate and adaptive immune responses. The effects of VP35 IRAD begin hours after infection and last for days. The VP24 IRAD affects a group of genes only partially overlapping that affected by the VP35 IRAD; in the hours after infection, it stimulates their expression, while later suppressing them. Overall, this study illustrates the global changes in the DC transcriptome associated with individual and combined effects of the VP35 and VP24 IRADs, which prevent maturation of DC and therefore make the T cell response less effective. The study also suggests the possibility of treatment of EBOV infection by targeting the identified suppressive pathways that could result in a functional adaptive immune response. Future studies should focus on an in-depth investigation of the role of IRADs in the deficient adaptive immune response and the disease caused by EBOV and on targeting the IRADs or unblocking their host target molecules to treat the infection.

ACKNOWLEDGMENTS

This study was supported by a John Sealy Memorial Endowment Fund pilot grant (Mechanisms of “Immune Paralysis” in Ebola Infections) (A.B.), a departmental startup grant from the University of Texas Medical Branch (A.B.), a McLaughlin Fellowship (M.L.), seed funds for pathogen genomics and other infectious disease and immunology initiatives involving next-generation sequencing from the UTMB Institute of Human Infections and Immunity (A.B.), and NIH grant U19 AI109945-01 (Molecular Basis for Ebola Virus Immune Paralysis) (A.B.).

We thank J. Towner and S. Nichol (CDC) for providing the EBOV full-length clone, Y. Kawaoka (University of Wisconsin) and H. Feldmann (NIH) for providing the EBOV NP, VP35, L, VP30, and T7 polymerase plasmids, and T. Garron for excellent technical assistance. We also thank Patrick Younan and Michelle Meyer for useful discussions of the data and critical readings of the manuscript.

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